AMPK promotes survival of c‐Myc‐positive melanoma cells by suppressing oxidative stress

Although c‐Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the Nras^Q61KINK4a^?/? mouse melanoma model to show that c‐Myc is essential for tumor initiation, maintenance, and metastasis. c‐Myc‐expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c‐Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c‐Myc‐positive melanoma cells activated and became dependent on the metabolic energy sensor AMP‐activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c‐Myc‐expressing melanoma cells, while AMPK activation protected against cell death of c‐Myc‐depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early‐stage human melanoma samples revealed an inverse correlation between C‐MYC and patient survival, suggesting that C‐MYC expression levels could serve as a prognostic marker for early‐stage disease.     

Distinct genomic traits of acral and mucosal melanomas revealed by targeted mutational profiling

The incidence of melanoma is rising globally including China. Comparing to Caucasians, the incidence of non‐cutaneous melanomas is significantly higher in Chinese. Herein, we performed genomic profiling of 89 Chinese surgically resected primary melanomas, including acral (n = 54), cutaneous (n = 22), and mucosal (n = 13), by hybrid capture‐based next‐generation sequencing. We show that mucosal melanomas tended to harbor more pathogenic mutations than other types of melanoma, though the biological significance of this finding remains uncertain. Chromosomal arm‐level alterations including 6q, 9p, and 10p/q loss were highly recurrent in all subtypes, but mucosal melanoma was significantly associated with increased genomic instability. Importantly, 7p gain significantly correlated with unfavorable clinical outcomes in non‐cutaneous melanomas, representing an intriguing prognostic biomarker of those subtypes. Furthermore, focal amplification of 4q12 (KIT, KDR, and PDGFRα) and RAD51 deletion were more abundant in mucosal melanoma, while NOTCH2 amplification was enriched in acral melanoma. Additionally, cutaneous melanomas had higher mutation load than acral melanomas, while mucosal melanomas did not differ from other subtypes in mutation burden. Together, our data revealed important features of acral and mucosal melanomas in Chinese including distinctive driver mutation pattern and increased genomic instability. These findings highlight the possibilities of combination therapies in the clinical management of melanoma.     

Met amplification and tumor progression in Cdkn2a‐deficient melanocytes

While many genetic alterations have been identified in melanoma, the relevant molecular events that contribute to disease progression are poorly understood. Most primary human melanomas exhibit loss of expression of the CDKN2A locus in addition to activation of the canonical mitogen‐activated protein kinase signaling pathway. In this study, we used a Cdkn2a‐deficient mouse melanocyte cell line to screen for secondary genetic events in melanoma tumor progression. Upon investigation, intrachromosomal gene amplification of Met, a receptor tyrosine kinase implicated in melanoma progression, was identified in Cdkn2a‐deficient tumors. RNA interference targeting Met in these tumor cells resulted in a significant delay in tumor growth in vivo compared with the control cells. MET expression is rarely detected in primary human melanoma but is frequently observed in metastatic disease. This study validates a role for Met activation in melanoma tumor progression in the context of Cdkn2a deficiency.     

Priming and filtering of antiherbivore defences among Nicotiana attenuata plants connected by mycorrhizal networks

Arbuscular mycorrhizal fungi (AMF) establish symbiotic associations with a majority of terrestrial plants to form underground common mycorrhizal networks (CMNs) that connect neighbouring plants. Because Nicotiana attenuata plants do not respond to herbivory‐elicited volatiles from neighbours, we used this ecological model system to evaluate if CMNs function in interplant transmission of herbivory‐elicited responses. A mesocosm system was designed to establish and remove CMNs linking N. attenuata plants to examine the herbivory‐elicited metabolic and hormone responses in CMNs‐connected “receiver” plants after the elicitation of “donor” plants by wounding (W) treated with Manduca sexta larval oral secretions (OS). AMF colonization increased constitutive jasmonate (JA and JA‐Ile) levels in N. attenuata roots but did not affect well‐characterized JAs‐regulated defensive metabolites in systemic leaves. Interestingly, larger JAs bursts, and higher levels of several amino acids and particular sectors of hydroxygeranyllinalool diterpene glycoside metabolism were elevated in the leaves of W + OS‐elicited “receivers” with CMN connections with “donors” that had been W + OS‐elicited 6 hr previously. Our results demonstrate that AMF colonization alone does not enhance systemic defence responses but that sectors of systemic responses in leaves can be primed by CMNs, suggesting that CMNs can transmit and even filter defence signalling among connected plants.     

Establishment and characterization of a novel ‘double‐hit’ follicular lymphoma cell line,FL‐SJC

About 5 per cent of follicular lymphoma (FL) cases are double‐hit (DH) lymphomas. Double‐hit follicular lymphoma (DHFL) cell lines can improve our understanding and drug development on FL. But there are only few DHFL cell lines. Here, we established a new MYC/BCL2 DHFL cell line, FL‐SJC. The cells were obtained from the hydrothorax of a patient with MYC/BCL2 DHFL and cultured for 140 passages in vitro. FL‐SJC cells demonstrated CD19⁺⁺, CD20⁺, CD22⁺⁺, HLA‐DR⁺, CD10⁺, CD38⁺, Lambda⁺ CD23^‐, CD5^‐ and Kappa^‐. The chromosome karyotypic analysis confirmed the co‐existence of t(8;22)(q24;q11) and t(14;18)(q32;q21), as well as additional abnormalities involving chromosomes 2 and 3. Fluorescence in situ hybridization analysis (FISH) showed IGH/BCL2 fusion gene and the MYC rearrangement. In addition, the FL‐SJC cells displayed KMT2D/MLL2 and CREBBP gene mutations. After subcutaneous inoculation of FL‐SJC cells, the SCID mice developed solid tumour masses within 6‐8 weeks. FL‐SJC cells were proven to be free of Epstein‐Barr (EB) virus infection and be multidrug‐resistant. In a conclusion, the FL‐SJC cell line has been identified as a novel MYC/BCL2 double‐hit follicular lymphoma that can be used as a potentially available tool for the clinical and basic research, together with the drug development for MYC/BCL2 DHFL.     

Genomic analysis and clinical management of adolescent cutaneous melanoma

Melanoma in young children is rare; however, its incidence in adolescents and young adults is rising. We describe the clinical course of a 15‐year‐old female diagnosed with AJCC stage IB non‐ulcerated primary melanoma, who died from metastatic disease 4 years after diagnosis despite three lines of modern systemic therapy. We also present the complete genomic profile of her tumour and compare this to a further series of 13 adolescent melanomas and 275 adult cutaneous melanomas. A somatic BRAF^V600E mutation and a high mutational load equivalent to that found in adult melanoma and composed primarily of C>T mutations were observed. A germline genomic analysis alongside a series of 23 children and adolescents with melanoma revealed no mutations in known germline melanoma‐predisposing genes. Adolescent melanomas appear to have genomes that are as complex as those arising in adulthood and their clinical course can, as with adults, be unpredictable.     

Near‐genomewide RNAi screening for regulators of BRAFV600E‐induced senescence identifies RASEF,a gene epigenetically silenced in melanoma

The activation of oncogenes in primary cells blocks proliferation by inducing oncogene‐induced senescence (OIS), a highly potent in vivo tumor‐suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near‐genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF^V600E‐induced arrest. With genome‐wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence‐associated (SA)‐β‐galactosidase activity, interleukins, and tumor suppressor p15^INK4B. Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.     

High frequency of PTEN mutations in nevi and melanomas from xeroderma pigmentosum patients

We examined nevi and melanomas in 10 xeroderma pigmentosum (XP) patients with defective DNA repair. The lesions had a lentiginous appearance with markedly increased numbers of melanocytes. Using laser capture microdissection, we performed DNA sequencing of 18 benign and atypical nevi and 75 melanomas (melanoma in situ and invasive melanomas). The nevi had a similar high frequency of PTEN mutations as melanomas [61% (11/18) versus 53% (39/73)]. Both had a very high proportion of UV‐type mutations (occurring at adjacent pyrimidines) [91% (10/11) versus 92% (36/39)]. In contrast to melanomas in the general population, the frequency of BRAF mutations (11%, 7/61), NRAS mutations (21%, 13/62), and KIT mutations (21%, 6/28) in XP melanomas was lower than for PTEN. Phospho‐S6 immunostaining indicated activation of the mTOR pathway in the atypical nevi and melanomas. Thus, the clinical and histological appearances and the molecular pathology of these UV‐related XP nevi and melanomas were different from nevi and melanomas in the general population.     

Wnt5A promotes an adaptive,senescent‐like stress response,while continuing to drive invasion in melanoma cells

We have previously shown that Wnt5A drives invasion in melanoma. We have also shown that Wnt5A promotes resistance to therapy designed to target the BRAF^V600E mutation in melanoma. Here, we show that melanomas characterized by high levels of Wnt5A respond to therapeutic stress by increasing p21 and expressing classical markers of senescence, including positivity for senescence‐associated β‐galactosidase (SA‐β‐gal), senescence‐associated heterochromatic foci (SAHF), H3K9Me chromatin marks, and PML bodies. We find that despite this, these cells retain their ability to migrate and invade. Further, despite the expression of classic markers of senescence such as SA‐β‐gal and SAHF, these Wnt5A‐high cells are able to colonize the lungs in in vivo tail vein colony‐forming assays. This clearly underscores the fact that these markers do not indicate true senescence in these cells, but instead an adaptive stress response that allows the cells to evade therapy and invade. Notably, silencing Wnt5A reduces expression of these markers and decreases invasiveness. The combined data point to Wnt5A as a master regulator of an adaptive stress response in melanoma, which may contribute to therapy resistance.     

MYC‐driven inhibition of the glutamate‐cysteine ligase promotes glutathione depletion in liver cancer

How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC‐driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl‐tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC‐driven tumors. We find that fewer glutamine‐derived carbons are incorporated into GSH in tumor tissue relative to non‐tumor tissue. Expression of GCLC, the rate‐limiting enzyme of GSH synthesis, is attenuated by the MYC‐induced microRNA miR‐18a. Inhibition of miR‐18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC‐driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC‐dependent attenuation of GCLC by miR‐18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.     

Rictor regulates the vasculogenic mimicry of melanoma via the AKT‐MMP‐2/9 pathway

Vasculogenic mimicry (VM)‐positive melanomas are usually associated with poor prognosis. Rictor, the key component of the rapamycin‐insensitive complex of mTOR (mTORC2), is up‐regulated in several cancers, especially in melanomas with poor prognosis. The aim of this study was to investigate the role of Rictor in the regulation of VM and the mechanism underlying this possible regulation. VM channels were found in 35 of 81 tested melanoma samples and high Rictor expression correlated with VM structures. Moreover, Kaplan–Meier survival curves indicated that VM structures and high Rictor expression correlated with shorter survival in patients with melanoma. In vitro, Rictor knockdown by short hairpin RNA (shRNA) significantly inhibited the ability of A375 and MUM‐2B melanoma cells to form VM structures, as evidenced by most tubes remaining open. Cell cycle analysis revealed that Rictor knockdown blocked cell growth and resulted in the accumulation of cells in G2/M phase, and cell migration and invasion were greatly affected after Rictor down‐regulation. Western blotting assays indicated that down‐regulating Rictor significantly inhibited the phosphorylation of AKT at Ser⁴⁷³ and Thr³⁰⁸, which subsequently inhibited the expression and activity of downstream MMP‐2/9, as confirmed by real‐time PCR and gelatin Zymography. MK‐2206, a small‐molecule inhibitor of AKT, similarly inhibited the activity of AKT and secretion of MMP‐2/9, further supporting that Rictor down‐regulation inhibits the phosphorylation of AKT and activity of downstream MMP‐2/9 to affect VM formation. In conclusion, Rictor plays an important role in melanoma VM via the Rictor—AKT—MMP‐2/9 signalling pathway.     

Metastatic risk and resistance to BRAF inhibitors in melanoma defined by selective allelic loss of ATG5

Melanoma is a paradigm of aggressive tumors with a complex and heterogeneous genetic background. Still, melanoma cells frequently retain developmental traits that trace back to lineage specification programs. In particular, lysosome-associated vesicular trafficking is emerging as a melanoma-enriched lineage dependency. However, the contribution of other lysosomal functions such as autophagy to melanoma progression is unclear, particularly in the context of metastasis and resistance to targeted therapy. Here we mined a broad spectrum of cancers for a meta-analysis of mRNA expression, copy number variation and prognostic value of 13 core autophagy genes. This strategy identified heterozygous loss of ATG5 at chromosome band 6q21 as a distinctive feature of advanced melanomas. Importantly, partial ATG5 loss predicted poor overall patient survival in a manner not shared by other autophagy factors and not recapitulated in other tumor types. This prognostic relevance of ATG5 copy number was not evident for other 6q21 neighboring genes. Melanocyte-specific mouse models confirmed that heterozygous (but not homozygous) deletion of Atg5 enhanced melanoma metastasis and compromised the response to targeted therapy (exemplified by dabrafenib, a BRAF inhibitor in clinical use). Collectively, our results support ATG5 as a therapeutically relevant dose-dependent rheostat of melanoma progression. Moreover, these data have important translational implications in drug design, as partial blockade of autophagy genes may worsen (instead of counteracting) the malignant behavior of metastatic melanomas.     

Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8+ T lymphocytes

CD8⁺ cytotoxic T lymphocytes (CTLs) are critical mediators of anti‐tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen‐presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c‐Met (c‐Met⁺ CTLs). Phenotypic and functional analysis of c‐Met⁺ CTLs reveals that they display enhanced cytolytic capacities compared to their c‐Met^? CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c‐Met⁺ CTLs in cell‐mediated cytotoxicity reactions in vitro and in vivo and abrogates T‐cell responses against metastatic melanoma in vivo. Finally, we establish in three murine tumor settings and in human melanoma tissues that c‐Met⁺ CTLs are a naturally occurring CD8⁺ T‐cell population. Together, our findings suggest that the HGF/c‐Met pathway could be exploited to control CD8⁺ T‐cell‐mediated anti‐tumor immunity.     

Protective role of mouse mast cell tryptase Mcpt6 in melanoma

Tryptase‐positive mast cells populate melanomas, but it is not known whether tryptase impacts on melanoma progression. Here we addressed this and show that melanoma growth is significantly higher in tryptase‐deficient (Mcpt6^?/?) versus wild‐type mice. Histochemical analysis showed that mast cells were frequent in the tumor stroma of both wild‐type and Mcpt6^?/? mice, and also revealed their presence within the tumor parenchyma. Confocal microscopy analysis revealed that tryptase was taken up by the tumor cells. Further, tryptase‐positive granules were released from mast cells and were widely distributed within the tumor tissue, suggesting that tryptase could impact on the tumor microenvironment. Indeed, gene expression analysis showed that the absence of Mcpt6 caused decreased expression of numerous genes, including Cxcl9, Tgtp2, and Gbp10, while the expression of 5p‐miR3098 was enhanced. The levels of CXCL9 were lower in serum from Mcpt6^?/? versus wild‐type mice. In further support of a functional impact of tryptase on melanoma, recombinant tryptase (Mcpt6) was taken up by cultured melanoma cells and caused reduced proliferation. Altogether, our results indicate a protective role of mast cell tryptase in melanoma growth.     

PTEN regulates IGF‐1R‐mediated therapy resistance in melanoma

Inhibition of the mitogen‐activated protein kinase (MAPK) pathway is a major advance in the treatment of metastatic melanoma. However, its therapeutic success is limited by the rapid emergence of drug resistance. The insulin‐like growth factor‐1 receptor (IGF‐1R) is overexpressed in melanomas developing resistance toward the BRAF^V600 inhibitor vemurafenib. Here, we show that hyperactivation of BRAF enhances IGF‐1R expression. In addition, the phosphatase activity of PTEN as well as heterocellular contact to stromal cells increases IGF‐1R expression in melanoma cells and enhances resistance to vemurafenib. Interestingly, PTEN‐negative melanoma cells escape IGF‐1R blockade by decreased expression of the receptor, implicating that only in melanoma patients with PTEN‐positive tumors treatment with IGF‐1R inhibitors would be a suitable strategy to combat therapy resistance. Our data emphasize the crosstalk and therapeutic relevance of microenvironmental and tumor cell‐autonomous mechanisms in regulating IGF‐1R expression and by this sensitivity toward targeted therapies.     

Frequent loss of heterozygosity in the β2-microglobulin region of chromosome 15 in primary human tumors

Downregulation or total loss of HLA class I expression on tumor cells is known as a mechanism of cancer immune escape. Alterations of the HLA phenotype are frequently due to mutations affecting genes encoding the HLA class I heavy chains located on chromosome 6p21 or the β2-microglobulin (β2m) gene encoding the light chain of the HLA complex located on chromosome 15q21. Frequently irreversible total loss of HLA class I molecules is due to the coincidence of two molecular events, the mutation of one β2m gene and the loss of the second copy. The latter is detectable as loss of heterozygosity (LOH) of microsatellite markers in the β2m region on chromosome 15q21 (LOH-15q21). Thus, LOH-15q21 might be an important event in the processes of HLA class I downregulation and total loss. Here we studied the frequency of LOH-15q21 in tumor tissues of different entities. By determining the status of heterozygosity of two microsatellite markers we detected LOH-15q21 in 44% of bladder carcinomas (n = 69), in 35% of colon carcinomas (n = 95), in 16% of melanomas (n = 70) but only in 7% of renal cancers (n = 45). Moreover, we observed a frequent coincidence of LOH-15q21 and LOH-6p21 in colorectal carcinoma, bladder carcinoma and melanoma, but not for renal carcinoma. We believe that the high incidence of LOH-15q21 in some malignancies and especially the coincidence of LOH-15q21 and LOH-6p21 might have a strong impact on tumor immunogenicity and on the efficiency of cancer immunotherapy.     

Mutations in KIT occur at low frequency in melanomas arising from anatomical sites associated with chronic and intermittent sun exposure

In melanoma, mutations in KIT are most frequent in acral and mucosal subtypes and rarely reported in cutaneous melanomas particularly those associated with intermittent UV exposure. Conversely melanomas arising within chronic sun damaged skin are considered to harbour KIT mutations at higher rates. To characterize the frequency of KIT mutations in a representative melanoma population, 261 patients from two Australian melanoma centres were prospectively screened for mutations in exons 11, 13 and 17 of the KIT gene. A total of 257 patients had cutaneous melanoma arising from non-acral sites and four were acral melanomas. No mucosal or ocular melanomas were analysed. KIT mutations were identified in five tumours (2% of the entire cohort) including two acral melanomas. Two of the three non-acral melanomas with KIT mutations were associated with markers of chronic sun damage as assessed by the degree of skin elastosis. In the remaining cohort, 43% had chronically sun damaged skin. This report confirms that within an Australian population, KIT mutations are infrequent in cutaneous melanomas associated with both intermittent and chronic sun exposed skin.